Project 1: Functional characterization of candidate cancer genes identified by genomic analysis of serous endometrial tumors During previous reporting periods, we published one of the first whole exome sequencing studies of serous endometrial tumors (Le Gallo et al., Nature Genetics 2012; 44:1310-5). The major finding of that study was our discovery that in serous endometrial tumors the FBXW7, CHD4, and SPOP genes are mutated at statistically significantly higher rates than the background mutation rate. This observation strongly suggests that somatic mutations in FBXW7, CHD4, and SPOP are pathogenic driver mutations that confer a selective advantage in serous endometrial tumorigenesis. However, proof of this hypothesis requires functional studies of the mutant proteins. Within the present reporting period we are continuing functional studies to determine how mutations in FBXW7 and SPOP, which both encode the substrate recognition components of ubiquitin ligase complexes, affect their function. In ongoing and planned studies that will extend into the next reporting period, we are characterizing the function of FBXW7 and SPOP mutants, using both biochemical and cell-based approaches. We are focusing our efforts on recurrent hotspot mutations that are present in endometrial carcinomas, but are rare or absent in other types of cancer, and which we therefore hypothesize are likely to be pathogenic driver mutations that contribute to endometrial tumorigenesis. Project 2. Identification of somatic mutations that underlie clear cell endometrial cancer During the previous reporting periods, we subjected DNAs from clear cell endometrial tumors and from the matched normal tissues to whole exome sequencing at the NIH Intramural Sequencing Center. We analyzed the exome sequencing data to identify somatic (tumor-specific) variants (present exclusively in the tumor DNA). We then prioritized a subset of nonsynonymously, somatically mutated genes, which we have deemed candidate cancer genes, for further downstream analysis. We Sanger sequenced these candidate cancer genes from another large cohort of clear cell ECs provided by extramural and international collaborators. In the past reporting period we: 1. Completed the mutation prevalence screen to attain high-level coverage or targeted genomic regions. 2. Nominated a subset of genes as novel pathogenic driver genes for clear cell EC 3. Prepared a manuscript describing our findings. In the incoming reporting period we will submit the manuscript to an international scientific journal and perform any additional experimentation that might be requested at peer-review. Project 3. Interrogation of somatic copy number alterations in serous and clear cell endometrial cancer In parallel to the mutational analyses described in Project 1 and 2, we are searching for somatic copy number alterations in serous and clear cell endometrial tumor genomes. In previous reporting periods, we used high-density SNP arrays to catalogue somatic genomic copy number alterations (gains and losses) that occur in serous and clear cell endometrial tumors. In the past reporting period, we prepared a draft manuscript describing some of our findings. In the incoming reporting period, we plan to complete the manuscript, submit to an international journal, and perform any additional experimentation that might be requested at peer-review. Collaborative projects Tumorigenesis is driven by genomic and epigenomic alterations, including altered methylation states. In the past, current and incoming reporting periods, we are collaborating with Dr. Laura Elnitski (NHGRI/NIH) to determine the genome-wide methylation states of endometrial carcinomas, including serous and clear cell subtypes.